Ptostaglandins are a group of lipid-soluble hormone mediators derived from the metabolism of arachidonic acid via the cyclooxygenase enzymatic pathway. In the prostaglandin biosynthetic pathway, arachidonic acid is first converted to the endoperoxide PGH2 by PGH2 synthases followed by the cell-specific isomerization or reduction of PGH2 to the active prostaglandins: PGD.sub.2, PGE.sub.2, PGF.sub.2.alpha., prostacyclin (PGI.sub.2) and thromboxane (TxA.sub.2). Following enzymatic conversion, prostaglandins exert their actions locally on the cells in which they were synthesized (autocrine) and/or on nearby cells (paracrine) through specific G protein-coupled receptors (Smith, (1992) Am. J. Physiol., 263: F181-F191) to either stimulate or inhibit the production of second messengers. Prostaglandins elicit a diverse spectrum of often opposing biological effects including muscle contraction and relaxation, platelet aggregation, vasodilation and inflammation.
PGE.sub.2 exhibits a broad range of actions in a number of tissues by binding to at least three EP receptor subtypes. It acts through pharmacologically distinct stimulatory (EP.sub.2) and inhibitory (EP.sub.3) PGE receptor subtypes to stimulate and inhibit cAMP formation, respectively (Sonnenburg, and Smith, (1988) J. Biol. Chem., 263: 6155-6160). PGE.sub.2 also stimulates calcium release and protein kinase C activity in the rabbit kidney collecting tubule, most likely by binding to the EP, receptor subtype that is coupled to stimulation of phospholipase C (Hebert et al., (1990) Am. J. Physiol., 259: F318-F325). The EP.sub.3 receptor subtype is involved in inhibition of gastric acid secretion, modulation of neurotransmitter release, inhibition of sodium and water reabsorption in the kidney tubule, potentiation of platelet aggregation at low concentrations (below 1 .mu.M) and inhibition of platelet aggregation at higher concentrations (Tynan et al., (1984) Prostaglandins, 27: 683-696; Matthews and Jones, (1993) British J. Pharmacol., 108: 363-369).
Development of therapeutic prostaglandins requires selective action at receptor subtypes. The murine EP.sub.2 and EP.sub.3 prostaglandin receptors have been cloned and sequenced (Honda et al., (1993) J. Biol. Chem., 268: 7759-7762; Sugimoto et al., (1992) J. Biol. Chem., 267: 6463-6466). The deduced protein sequences indicate that both are members of the G protein-linked receptor superfamily, having seven putative membrane-spanning hydrophobic domains. The proteins share significant amino acid sequence similarity with other members of this family including the thromboxane (TP) receptor (Hirata et al., (1991) Nature 349: 617-620), rhodopsin and the adrenergic receptors. In order to characterize the pharmacology of the murine EP.sub.3 receptor, the gene was transfected into COS-7 cells which lack the EP.sub.3 receptor and competition binding assays using tritiated PGE.sub.2 were performed on the plasma membrane fraction (Sugimoto et al., (1992) J. Biol. Chem., 267: 6463-6466).
However, these results only addressed the binding of compounds to the murine receptors. There is still the need to identify compounds which specifically bind to the human EP.sub.3 receptor, since the pharmacology of rodent G-protein coupled receptors does not always match their human homologs (Oksenberg et al., (1992) Nature, 360: 161-163; Link et al., (1992) Mol. Pharmacol., 42: 16-27).